Sirtuin 5 depletion impairs mitochondrial function in human proximal tubular epithelial cells

Ischemia is a major cause of kidney damage. Proximal tubular epithelial cells (PTECs) are highly susceptible to ischemic insults that frequently cause acute kidney injury (AKI), a potentially life-threatening condition with high mortality. Accumulating evidence has identified altered mitochondrial function as a central pathologic feature of AKI. The mitochondrial NAD+-dependent enzyme sirtuin 5 (SIRT5) is a key regulator of mitochondrial form and function, but its role in ischemic renal injury (IRI) is unknown. SIRT5 expression was increased in murine PTECs after IRI in vivo and in human PTECs (hPTECs) exposed to an oxygen/nutrient deprivation (OND) model of IRI in vitro. SIRT5-depletion impaired ATP production, reduced mitochondrial membrane potential, and provoked mitochondrial fragmentation in hPTECs. Moreover, SIRT5 RNAi exacerbated OND-induced mitochondrial bioenergetic dysfunction and swelling, and increased degradation by mitophagy. These findings suggest SIRT5 is required for normal mitochondrial function in hPTECs and indicate a potentially important role for the enzyme in the regulation of mitochondrial biology in ischemia.


Supplementary Material
Methods In vitro ischemia. HKC-8 cells (hPTECs) were seeded in 6-well plates (Corning) at a density of 6x10 5 cells/well. After 24 hours, cells were 90% confluent and were used for experiments. To mimic ischemia in vitro a combined oxygen and nutrient-deprivation (OND) model was developed, which implements both aspects of ischemia in vivo i.e. hypoxia and nutrient starvation. To differentiate between the impact of (i) oxygen deprivation (OD; hypoxia) and (ii) nutrient deprivation (ND; starvation), hPTECs were For WB, fluorescence-activated cell sorting (FACS) and mitochondrial structure analyses (confocal and transmission electron microscopy (TEM)), hPTECs were seeded in T25 flasks (Corning) at a density of 1.5x10 6 cells/flask and transfected the following day.
Twenty-four hours post-transfection, transfection medium was changed to CM to reduce the cytotoxicity of the transfection reagent. Forty-eight hours post-transfection, cells were re-plated in 6-well plates (WB and FACS analyses; 6x10 5 cells/well), 8-well chamber slides (Nunc Lab-Tek) (confocal microscopy; 5x10 4  Ultrastructural analysis of mitochondria. hPTECs (control RNAi-and SIRT5 RNAitreated) were seeded in 10cm petri dishes (Corning) at a density of 4.5x10 6 cells/plate.
The following day, cells underwent either OND or were cultured in CM under normoxic conditions (control; protocol described in "In vitro ischemia") for 6 hours. Cells were washed with PBS (gibco), detached from the culture plates using 3ml 0.05% Trypsin-EDTA (gibco) and suspended in 7ml CM for trypsin inactivation. Cells were centrifuged for 5 minutes at 400xg at room temperature (RT), supernatants removed and the pellets washed with PBS. After centrifugation for 5 minutes at 400xg, supernatants were aspirated and cells fixed in 5ml 2.5% glutaraldehyde buffered with 100mM sodium cacodylate (pH 7.2; agar scientific) for 24 hours at RT and stored for up to a week at 4ºC. Sample processing and imaging were performed by the Histopathology Department (Great Ormond Street Hospital, London). Briefly, after secondary fixation in 1% osmium tetroxide (agar scientific) for 2 hours, samples were dehydrated in graded ethanols, transferred to propylene oxide (agar scientific) then infiltrated and embedded in Agar 100 epoxy resin (agar scientific). Polymerisation was carried out at 60°C for 48 hours. Ultrathin sections (90nm) were cut using a Diatome diamond knife on a Leica EM UC7 ultramicrotome.
Sections were collected on copper grids and stained with alcoholic uranyl acetate (agar scientific) and lead citrate (agar scientific). Sections were examined with a JEOL 1400 TEM and digital images recorded using an AMT XR80 digital camera.
Assessment of mitochondrial function using respirometry. Oxygen consumption rate (OCR; a measure of mitochondrial function) and extracellular acidification rate (ECAR; a measure of glycolysis) were assessed using a Seahorse XFp Analyzer (Agilent). hPTECs (control RNAi-and SIRT5 RNAi-treated) were seeded in Seahorse XFp 8-well plates in CM and grown overnight. Cells were exposed to 6 hours OND. After OND, HBSS was replaced by HEPES-buffered DMEM (Agilent) and plates were incubated in a humidified chamber without CO2 for 1 hour at 37°C to stabilize pH and temperature. Sensor cartridges (Agilent) required for mitochondrial toxin injection were hydrated the day before the assay and kept in a humidified incubator without CO2 at 37°C. After assessment of basal respiration, oligomycin (2μM; a mitochondrial ATP-synthase inhibitor; gives insight into proton leak), Carbonyl cyanide-4-phenylhydrazone (FCCP) (0.75μM; a mitochondrial uncoupler; reveals maximal cellular respiration) and rotenone (1μM) with antimycin A (1μM; electron transport chain complex 1 and 3 inhibitors; inhibition of mitochondrial respiration to analyze non-mitochondrial respiration) were sequentially injected.
Measurement of OCR and ECAR was carried out every 3 minutes for 2 hours. OCR and ECAR were normalized to total cell number/well (CyQUANT; Life Technologies). Data were analysed using the Wave software (Agilent).
Crude mitochondrial isolation. Kidneys from WT and Sirt5 -/mice were decapsulated, chopped into small pieces (1mm 3 ), transferred to pre-cooled glass tubes and isotonic homogenization buffer (0.32M sucrose, 1mM EDTA, 10mM Tris-HCl; pH 7.4) was added (total volume of 2ml). Tissues were homogenized using a Potter Elvehejm tissue homogenizer (Eurostar). The homogenate was transferred to a 50ml centrifuge tube (Corning) and centrifuged at 1500xg for 10min at 4ºC. The mitochondria-containing supernatant was transferred to a new 50ml tube and centrifuged at 11,500xg for 10min at 4ºC. The supernatant was removed and the pellets re-suspended in 500µl homogenization buffer. Crude mitochondrial extracts were flash-frozen in liquid N2 and stored at -80ºC until analyzed.
Complex II activity assay. Succinate ubiquinone oxidoreductase (complex II) activity was measured in crude mitochondrial extracts from WT and Sirt5 -/kidneys. A modified version based on the method of Hatefi and Stiggall 1 was used. This assay measures the succinate-ubiquinone reductase activity indirectly through the CoQ2 (ubiquinone-2)dependent reduction of 2,6-dichloroindophenol (DCIP), a blue dye which shows maximal absorption at 600nm. The time-dependent decrease in DCIP absorption allows the determination of complex II activity. Before complex II activity could be assessed, mitochondrial extracts were subjected to three freeze-thaw cycles to release mitochondrial enzymes: Samples were flash-frozen in liquid nitrogen and subsequently thawed in a water-bath at 30°C for 2 minutes.
To determine complex II activity, absorption was determined in a test cuvette and compared to the absorption of a reference cuvette. An assay solution was prepared: potassium phosphate buffer (50mM; K2HPO4; KH2PO4), EDTA (100µM), DCIP (74µM), sodium-succinate (20mM), sodium-cyanide (1mM; complex IV inhibitor which prevents electron flow to O2), rotenone (10µM; complex I inhibitor which prevents electron flow along complex I). The reference cuvette contained the assay solution and in addition, the complex II inhibitor 2-thenoyltrifluoroacetone (TTFA; 1mM) and the sample (shows succinate-dependent, CoQ2-independent reduction of DCIP). The test cuvette also contained the assay buffer. Sequential addition of sample, CoQ2 and TTFA allowed determination of complex II activity. The detailed process was carried out as follows: (1) Assay solution was added to the cuvette. (2) At t=50s, sample was added to the assay solution, mixed carefully and absorption was measured at t=120s and t=180s to determine the succinate-dependent, but CoQ2-independent reduction of DCIP (background). A Hitachi U-3310 UV-Vis spectrophotometer was used to measure absorption of DCIP. All measurements were carried out in quadruplicate at 30°C. Complex II enzyme activities were normalised to the total protein content of the sample as determined by the DC protein assay (Bio-Rad).
Seventy-two hours post-transfection cells were exposed to 6 hours of OND (1%O2+HBSS) or normoxia and complete medium (21%O2+CM). Integrated optical density (IOD) of Mtophagy dye was assessed using Image-Pro Premier 10 (Media Cybernetics). Data are from two independent experiments and a total of >40 cells were analysed/condition. Data are median ± IQR. To determine statistical significance a Kruskal-Wallis test was carried out followed by Dunn's post hoc test to normalize for multiple comparisons. *p<0.05 and ****p<0.0001.